Eukaryotic translation elongation is a determinant of fidelity and processivity during protein synthesis. The regulation of this process is largely based on the functional relationship between eukaryotic Elongation Factor 1A (eEF1A) and its cofactors the guanine nucleotide exchange factor eEF1Ba, aminoacyl-tRNA (aa-tRNA), the fungal specific elongation factor eEF3, and the actin cytoskeleton. eEF1A is the GTP binding protein that delivers aa-tRNA to the ribosomal A-site. eEF1A requires the guanine nucleotide exchange factor (GEF) eEF1Ba? for reactivation to the GTP-bound form. eEF3 is an essential fungal-specific elongation factor that has a functional and physical interaction with eEF1A. eEF1A further interacts with proteins outside the translational apparatus, in particular actin and actin binding proteins. Evidence in multiple eukaryotic systems points to a functional role of the cytoskeleton in translational regulation, where the eEF1A-actin interaction likely plays a key role. The unifying thread of this proposal is the hypothesis that the functional interplay between eEF1A, its co-factors in protein synthesis (aa-tRNA, eEF1Ba, and eEF3), and actin is a key regulatory and communication point between protein synthesis and the cytoskeleton. The distribution of eEF1A between these roles would subsequently affect the regulation of gene expression. Aim 1 is based on our hypothesis that eEF1A is a member of the actin bundling protein family that performs a role unrelated to its elongation activity but linked through the actin cytoskeleton to translation initiation. Aim 2 is based on our hypothesis that in addition to its role as a guanine nucleotide exchange factor, eEF1Ba helps facilitate aa-tRNA binding to the GTP-bound form of eEF1A and thus competes with actin for binding to eEF1A. Aim 3 is based on our data on the eEF3-eEF1A interaction that supports our model that eEF3 binding to eEF1A is mutually exclusive to actin binding. Furthermore, the crystal structure of eEF3 and cryo-EM reconstitution bound to the 80S yeast ribosome led to the hypothesis that the chromodomain and the ABC2 domain are essential for the function of eEF3 on the ribosome and its effects on translational fidelity at the A-site. Overall, the work will serve as a model for elongation factor function in human systems regarding the unique role of eEF1A in coordinating the interplay between translation elongation and the cytoskeleton. Further, this will shed light on the functional consequences of altered eEF1A levels and activities demonstrated to occur in different physiological conditions and disease states. In addition, our increased knowledge of fungal translation elongation can serve as a platform for the future targeting of the fungal-specific aspects of this process, such as eEF3, for the development of new antifungal treatments. With the yeast Saccharomyces cerevisiae, the progress in the prior funding period can now be utilized for an integrated genetic, biochemical and cell biological analysis on the functional roles, physical states and unique aspects of eEF1A and its associated factors in the context of regulated of gene expression.